Measurement of left ventricular volume with admittance incorporated onto percutaneous ventricular assist device.

Percutaneous ventricular assist devices (pVADs) incorporated with admittance electrodes have been validated in animal studies for accurate instantaneous volumetric measurements. Since miniaturization of the pVAD profile is a priority to reduce vascular complications in patients, our study aimed to validate admittance measurements using three electrodes instead of the standard four. Complex admittance was measured between an electrode pair and a pVAD metallic blood-intake tip, both with finite element analysis and on the benchtop. The catheter and electrode arrays were first simulated inside prolate ellipsoid models of the left ventricle (LV) demonstrating current flow throughout all parts of the LV as well as minimal influence of off-center catheter placement in the recorded signal. Admittance measurements were validated in 3D-printed models of healthy and dilated hearts (100-400 mL end-diastolic volumes). Minimal interference between a pVAD motor and the current signal of our admittance system was demonstrated. A modified Wei's equation focused on three electrodes was developed to be compatible with reduced profile pVADs occurring clinically, incorporated with admittance electrodes and wires. The modified equation was compared against Wei's original equation showing improved accuracy of calculated volumes. Reducing electrode footprint can simplify the incorporation of Admittance technology on any pVAD, allowing for instantaneous recognition of native heart recovery and assistance with pVAD weaning.

Genomic Characterization of Highly Pathogenic Avian Influenza A H5N1 Virus Newly Emerged in Dairy Cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in United States for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post-infection, highlighting the need for continued surveillance to monitor public health threats.

A causal machine-learning framework for studying policy impact on air pollution: A case-study in COVID-19 lockdowns.

When studying the impact of policy interventions or natural experiments on air pollution, such as new environmental policies and opening or closing an industrial facility, careful statistical analysis is needed to separate causal changes from other confounding factors. Using COVID-19 lockdowns as a case-study, we present a comprehensive framework for estimating and validating causal changes from such perturbations. We propose using flexible machine learning-based comparative interrupted time series (CITS) models for estimating such a causal effect. We outline the assumptions required to identify causal effects, showing that many common methods rely on strong assumptions that are relaxed by machine learning models. For empirical validation, we also propose a simple diagnostic criterion, guarding against false effects in baseline years when there was no intervention. The framework is applied to study the impact of COVID-19 lockdowns on NO2 in the eastern US. The machine learning approaches guard against false effects better than common methods and suggest decreases in NO2 in Boston, New York City, Baltimore, and Washington D.C. The study showcases the importance of our validation framework in selecting a suitable method and the utility of a machine learning based CITS model for studying causal changes in air pollution time series.

Sialic Acid Receptor Specificity in Mammary Gland of Dairy Cattle Infected with Highly Pathogenic Avian Influenza A(H5N1) Virus.

In March 2024, the US Department of Agriculture's Animal and Plant Health Inspection Service reported detection of highly pathogenic avian influenza (HPAI) A(H5N1) virus in dairy cattle in the United States for the first time. One factor that determines susceptibility to HPAI H5N1 infection is the presence of specific virus receptors on host cells; however, little is known about the distribution of the sialic acid (SA) receptors in dairy cattle, particularly in mammary glands. We compared the distribution of SA receptors in the respiratory tract and mammary gland of dairy cattle naturally infected with HPAI H5N1. The respiratory and mammary glands of HPAI H5N1-infected dairy cattle are rich in SA, particularly avian influenza virus-specific SA α2,3-gal. Mammary gland tissues co-stained with sialic acids and influenza A virus nucleoprotein showed predominant co-localization with the virus and SA α2,3-gal. HPAI H5N1 exhibited epitheliotropism within the mammary gland, and we observed rare immunolabeling within macrophages.

Associations of Urban Blue and Green Spaces With Coronary Artery Calcification in Black Individuals and Disadvantaged Neighborhoods.

BACKGROUND: Proximity to urban blue and green spaces has been associated with improved cardiovascular health; however, few studies have examined the role of race and socioeconomic status in these associations. METHODS: Data were from the CARDIA study (Coronary Artery Risk Development in Young Adults). We included longitudinal measurements (1985-1986 to 2010-2011) of blue and green spaces, including percentage of blue space cover, distance to the nearest river, green space cover, and distance to the nearest major park. Presence of coronary artery calcification (CAC) was measured with noncontrast cardiac computed tomography in 2010 to 2011. The associations of blue and green spaces with CAC were assessed with generalized estimating equation regression with adjustment for demographics, individual and neighborhood socioeconomic status, health-related behaviors, and other health conditions. We conducted stratified analyses by race and neighborhood deprivation score to investigate whether the association varied according to social determinants of health. RESULTS: The analytic sample included 1365 Black and 1555 White participants with a mean±SD age of 50.1±3.6 years. Among Black participants, shorter distance to a river and greater green space cover were associated with lower odds of CAC (per interquartile range decrease [1.45 km] to the river: odds ratio [OR], 0.90 [95% CI, 0.84-0.96]; per 10%-point increase of green space cover: OR, 0.85 [95% CI, 0.75-0.95]). Among participants in deprived neighborhoods, greater green space cover was associated with lower odds of CAC (per a 10%-point increase: OR, 0.89 [95% CI, 0.80-0.99]), whereas shorter distance to the park was associated with higher odds of CAC (per an interquartile range decrease [5.3 km]: OR, 1.07 [95% CI, 1.00-1.15]). Black participants in deprived neighborhoods had lower odds of CAC with shorter distance to a river (per an interquartile range decrease: OR, 0.90 [95% CI, 0.82-0.98]) and greater green space cover (per a 10%-point increase: OR, 0.85 [95% CI, 0.75-0.97]). There was no statistical interaction between the blue and green spaces and race or neighborhood characteristics in association with CAC. CONCLUSIONS: Longitudinally, shorter distance to a river and greater green space cover were associated with less CAC among Black participants and those in deprived neighborhoods. Shorter distance to a park was associated with increased odds of CAC among participants in deprived neighborhoods. Black participants residing in more deprived neighborhoods showed lower odds of CAC in association with greater exposure to river and green space cover.

A simplified method for preventing post-mortem alterations of brain prostanoids for true in situ level quantification.

Dramatic post-mortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing post-mortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PG are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent post-mortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100 °C after ∼140 sec during boiling, though 3 min boiling was required to completely prevent post-mortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant post-mortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents post-mortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Diverse Gene Regulatory Mechanisms Alter Rattlesnake Venom Gene Expression at Fine Evolutionary Scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

Tofacitinib uptake by patient-derived intestinal organoids predicts individual clinical responsiveness.

Background & Aims: Despite increasing therapeutic options in the treatment of ulcerative colitis (UC), achieving disease remission remains a major clinical challenge. Nonresponse to therapy is common and clinicians have little guidance in selecting the optimal therapy for an individual patient. This study examined whether patient-derived materials could predict individual clinical responsiveness to the Janus kinase (JAK) inhibitor, tofacitinib, prior to treatment initiation. Method: In 48 patients with UC initiating tofacitinib, we longitudinally collected clinical covariates, stool, and colonic biopsies to analyze the microbiota, transcriptome, and exome variations associated with clinical responsiveness at week 24. We established patient-derived organoids (n = 23) to determine how their viability upon stimulation with proinflammatory cytokines in the presence of tofacitinib related to drug responsiveness in patients. We performed additional biochemical analyses of organoids and primary tissues to identify the mechanism underlying differential tofacitinib sensitivity. Results: The composition of the gut microbiota, rectal transcriptome, inflammatory biomarkers, and exome variations were indistinguishable among UC patients prior to tofacitinib treatment. However, a subset of patient-derived organoids displayed reduced sensitivity to tofacitinib as determined by the ability of the drug to inhibit STAT1 phosphorylation and loss of viability upon cytokine stimulation. Remarkably, sensitivity of organoids to tofacitinib predicted individual clinical patient responsiveness. Reduced responsiveness to tofacitinib was associated with decreased levels of the cationic transporter MATE1, which mediates tofacitinib uptake. Conclusions: Patient-derived intestinal organoids predict and identify mechanisms of individual tofacitinib responsiveness in UC. Specifically, MATE1 expression predicted clinical response to tofacitinib.

Nitrogen dioxide exposure, health outcomes, and associated demographic disparities due to gas and propane combustion by U.S. stoves.

Gas and propane stoves emit nitrogen dioxide (NO2) pollution indoors, but the exposures of different U.S. demographic groups are unknown. We estimate NO2 exposure and health consequences using emissions and concentration measurements from >100 homes, a room-specific indoor air quality model, epidemiological risk parameters, and statistical sampling of housing characteristics and occupant behavior. Gas and propane stoves increase long-term NO2 exposure 4.0 parts per billion volume on average across the United States, 75% of the World Health Organization's exposure guideline. This increased exposure likely causes ~50,000 cases of current pediatric asthma from long-term NO2 exposure alone. Short-term NO2 exposure from typical gas stove use frequently exceeds both World Health Organization and U.S. Environmental Protection Agency benchmarks. People living in residences <800 ft2 in size incur four times more long-term NO2 exposure than people in residences >3000 ft2 in size; American Indian/Alaska Native and Black and Hispanic/Latino households incur 60 and 20% more NO2 exposure, respectively, than the national average.

Phylogeny and historical biogeography of the swallow family (Hirundinidae) inferred from comparisons of thousands of UCE loci.

Swallows (Hirundinidae) are a globally distributed family of passerine birds that exhibit remarkable similarity in body shape but tremendous variation in plumage, sociality, nesting behavior, and migratory strategies. As a result, swallow species have become models for empirical behavioral ecology and evolutionary studies, and variation across the Hirundinidae presents an excellent opportunity for comparative analyses of trait evolution. Exploiting this potential requires a comprehensive and well-resolved phylogenetic tree of the family. To address this need, we estimated swallow phylogeny using genetic data from thousands of ultraconserved element (UCE) loci sampled from nearly all recognized swallow species. Maximum likelihood, coalescent-based, and Bayesian approaches yielded a well-resolved phylogenetic tree to the generic level, with minor disagreement among inferences at the species level, which likely reflect ongoing population genetic processes. The UCE data were particularly useful in helping to resolve deep nodes, which previously confounded phylogenetic reconstruction efforts. Divergence time estimates from the improved swallow tree support a Miocene origin of the family, roughly 13 million years ago, with subsequent diversification of major groups in the late Miocene and Pliocene. Our estimates of historical biogeography support the hypothesis that swallows originated in the Afrotropics and have subsequently expanded across the globe, with major in situ diversification in Africa and a secondary major radiation following colonization of the Neotropics. Initial examination of nesting and sociality indicates that the origin of mud nesting - a relatively rare nest construction phenotype in birds - was a major innovation coincident with the origin of a clade giving rise to over 40% of extant swallow diversity. In contrast, transitions between social and solitary nesting appear less important for explaining patterns of diversification among swallows.

Integrating population genetics, stem cell biology and cellular genomics to study complex human diseases.

Human pluripotent stem (hPS) cells can, in theory, be differentiated into any cell type, making them a powerful in vitro model for human biology. Recent technological advances have facilitated large-scale hPS cell studies that allow investigation of the genetic regulation of molecular phenotypes and their contribution to high-order phenotypes such as human disease. Integrating hPS cells with single-cell sequencing makes identifying context-dependent genetic effects during cell development or upon experimental manipulation possible. Here we discuss how the intersection of stem cell biology, population genetics and cellular genomics can help resolve the functional consequences of human genetic variation. We examine the critical challenges of integrating these fields and approaches to scaling them cost-effectively and practically. We highlight two areas of human biology that can particularly benefit from population-scale hPS cell studies, elucidating mechanisms underlying complex disease risk loci and evaluating relationships between common genetic variation and pharmacotherapeutic phenotypes.

Chemotherapy-induced acceleration of DNA methylation-based biological age in breast cancer.

Breast cancer is the most common cancer diagnosed in women and is often treated with chemotherapy. Although previous studies have demonstrated increasing biological age in patients who receive chemotherapy, evaluation of this association with DNA methylation-based markers of biological ageing may provide novel insight into the role of chemotherapy on the ageing process. We therefore sought to investigate the association between chemotherapy and markers of biological ageing as estimated from DNA methylation in women with breast cancer. DNA methylation profiling was performed on peripheral blood collected from 18 patients before and after the first cycle of chemotherapy using the Infinium HumanMethylation450 BeadChip. Six markers of biological age acceleration were estimated from DNA methylation levels. Multiple linear regression analyses were performed to evaluate the association between each metric of biological age acceleration and chemotherapy. After adjusting for chronological age and race, intrinsic epigenetic age acceleration (p = 0.041), extrinsic epigenetic age acceleration (p = 0.050), PhenoAge acceleration (p = 0.001), GrimAge acceleration (p < 0.001), and DunedinPACE (p = 0.006) were significantly higher and telomere length (p = 0.027) was significantly lower following the first cycle of chemotherapy compared to before treatment initiation. These results demonstrate greater biological ageing as estimated from DNA methylation following chemotherapy in women with breast cancer. Our findings illustrate that cytotoxic therapies may modulate the ageing process among breast cancer patients and may also have implications for age-related health conditions in cancer survivors.

Microbiota and metabolic adaptation shape Staphylococcus aureus virulence and antimicrobial resistance during intestinal colonization.

Depletion of microbiota increases susceptibility to gastrointestinal colonization and subsequent infection by opportunistic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). How the absence of gut microbiota impacts the evolution of MRSA is unknown. The present report used germ-free mice to investigate the evolutionary dynamics of MRSA in the absence of gut microbiota. Through genomic analyses and competition assays, we found that MRSA adapts to the microbiota-free gut through sequential genetic mutations and structural changes that enhance fitness. Initially, these adaptations increase carbohydrate transport; subsequently, evolutionary pathways largely diverge to enhance either arginine metabolism or cell wall biosynthesis. Increased fitness in arginine pathway mutants depended on arginine catabolic genes, especially nos and arcC, which promote microaerobic respiration and ATP generation, respectively. Thus, arginine adaptation likely improves redox balance and energy production in the oxygen-limited gut environment. Findings were supported by human gut metagenomic analyses, which suggest the influence of arginine metabolism on colonization. Surprisingly, these adaptive genetic changes often reduced MRSA's antimicrobial resistance and virulence. Furthermore, resistance mutation, typically associated with decreased virulence, also reduced colonization fitness, indicating evolutionary trade-offs among these traits. The presence of normal microbiota inhibited these adaptations, preserving MRSA's wild-type characteristics that effectively balance virulence, resistance, and colonization fitness. The results highlight the protective role of gut microbiota in preserving a balance of key MRSA traits for long-term ecological success in commensal populations, underscoring the potential consequences on MRSA's survival and fitness during and after host hospitalization and antimicrobial treatment.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

Chemosensory detection of polyamine metabolites guides C. elegans to nutritive microbes.

Much is known about molecular mechanisms by which animals detect pathogenic microbes, but how animals sense beneficial microbes remains poorly understood. The roundworm Caenorhabditis elegans is a microbivore that must distinguish nutritive microbes from pathogens. We characterized a neural circuit used by C. elegans to rapidly discriminate between nutritive bacteria and pathogens. Distinct sensory neuron populations responded to chemical cues from nutritive Escherichia coli and pathogenic Enterococcus faecalis, and these neural signals are decoded by downstream AIB interneurons. The polyamine metabolites cadaverine, putrescine, and spermidine produced by E. coli activate this neural circuit and elicit positive chemotaxis. Our study shows how polyamine odorants can be sensed by animals as proxies for microbe identity and suggests that, hence, polyamines might have widespread roles brokering host-microbe interactions.

DELE1 promotes translation-associated homeostasis, growth, and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1's protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Patient Subtyping Analysis of Baseline Multi-omic Data Reveals Distinct Pre-immune States Predictive of Vaccination Responses.

Understanding the molecular mechanisms that underpin diverse vaccination responses is a critical step toward developing efficient vaccines. Molecular subtyping approaches can offer valuable insights into the heterogeneous nature of responses and aid in the design of more effective vaccines. In order to explore the molecular signatures associated with the vaccine response, we analyzed baseline transcriptomics data from paired samples of whole blood, proteomics and glycomics data from serum, and metabolomics data from urine, obtained from influenza vaccine recipients (2019-2020 season) prior to vaccination. After integrating the data using a network-based model, we performed a subtyping analysis. The integration of multiple data modalities from 62 samples resulted in five baseline molecular subtypes with distinct molecular signatures. These baseline subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation across subtypes in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these significant differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining vaccine response and efficacy. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

Patterns of genetic divergence in the Rio Grande cooter (Pseudemys gorzugi), a riverine turtle inhabiting an arid and anthropogenically modified system.

The lower Rio Grande and Pecos River of the southwest United States have been heavily modified by human activities, profoundly impacting the integrity of their aquatic wildlife. In this context, we focused our study on the population genomics of the Rio Grande Cooter (Pseudemys gorzugi), a freshwater turtle of increasing conservation concern, residing in these two rivers and their tributaries. The genetic data revealed two distinct populations: one in the Pecos and Black Rivers of New Mexico and another in the Rio Grande and Devils River of Texas, with admixed individuals identified at the confluence of the Rio Grande and Pecos River. In addition to having a smaller geographic range, we found lower observed heterozygosity, reduced nucleotide diversity, and a smaller effective population size (Ne) in New Mexico population. Our results depict a significant isolation-by-distance pattern across their distribution, with migration being notably infrequent at river confluences. These findings are pivotal for future conservation and restoration strategies, emphasizing the need to recognize the unique needs of each population.

Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.

Total organic carbon measurements reveal major gaps in petrochemical emissions reporting.

Anthropogenic organic carbon emissions reporting has been largely limited to subsets of chemically speciated volatile organic compounds. However, new aircraft-based measurements revealed total gas-phase organic carbon emissions that exceed oil sands industry-reported values by 1900% to over 6300%, the bulk of which was due to unaccounted-for intermediate-volatility and semivolatile organic compounds. Measured facility-wide emissions represented approximately 1% of extracted petroleum, resulting in total organic carbon emissions equivalent to that from all other sources across Canada combined. These real-world observations demonstrate total organic carbon measurements as a means of detecting unknown or underreported carbon emissions regardless of chemical features. Because reporting gaps may include hazardous, reactive, or secondary air pollutants, fully constraining the impact of anthropogenic emissions necessitates routine, comprehensive total organic carbon monitoring as an inherent check on mass closure.